The role of DNA methylation in ovarian cancer chemoresistance: A narrative review

Abstract Background and Aims Ovarian cancer (OC) is the most lethal gynecological cancer. In 2018, it was responsible for over 180,000 deaths worldwide. The high mortality rate is the culmination of a lack of early diagnosis and high rates of chemotherapy resistance, which is synonymous with disease recurrence. Over the last two decades, an increasingly significant role of epigenetic mechanisms, in particular DNA methylation, has emerged. This review will discuss several of the most significant genes whose hypo/hypermethylation profiles are associated with chemoresistance. Aside from functionally elucidating and evaluating these epimutations, this review will discuss recent trials of DNA methyltransferase inhibitors (DNMTi). Finally, we will propose future directions that could enhance the feasibility of utilizing these candidate epimutations as clinical biomarkers. Methods To perform this review, a comprehensive literature search based on our keywords was conducted across the online databases PubMed and Google Scholar for identifying relevant studies published up until August 2022. Results Epimutations affecting MLH1, MSH2, and Ras‐association domain family 1 isoform A (DNA damage repair and apoptosis); ATP‐binding cassette subfamily B member 1 and methylation‐controlled J (drug export); secreted frizzled‐related proteins (Wnt/β‐catenin signaling), neurocalcin delta (calcium and G protein‐coupled receptor signaling), and zinc finger protein 671 all have potential as biomarkers for chemoresistance. However, specific uncertainties relating to these epimutations include histotype‐specific differences, intrinsic versus acquired chemoresistance, and the interplay with complete surgical debulking. DNMTi for chemoresistant OC patients has shown some promise; however, issues surrounding their efficacy and dose‐limiting toxicities remain; a personalized approach is required to maximize their effectiveness. Conclusion Establishing a panel of aberrantly methylated chemoresistance‐related genes to predict chemoresponsiveness and patients' suitability to DNMTi could significantly reduce OC recurrence, while improving DNMTi therapy viability. To achieve this, a large‐scale prospective genome‐wide DNA methylation profile study that spans different histotypes, includes paired samples (before and after chemotherapy), and integrates transcriptomic and methylomic analysis, is warranted.


| INTRODUCTION
Over the last three decades, there has been an average increase in 5-year survival rates of 20% across all cancers. 1 However, such improvements have not been observed in ovarian cancer (OC): this "silent killer" remains the most lethal gynecological cancer with a 5-year survival rate of 45%. 2 Epithelial ovarian cancer (EOC) is the most common type, constituting 85% of all cases and comprising four main histotypes: serous, endometrioid, clear-cell, and mucinous. 3 EOC has traditionally been treated through surgical tumor debulking followed by combined platinum/taxane therapy-primarily carboplatin and paclitaxel. In cases of advanced, platinum-sensitive OC, recent NICE guidance has approved the use of Olaparib (PARP inhibitor) alongside bevacizumab for treating tumors that exhibit homologous recombination deficiency. 4 The utility of Olaparib as a maintenance therapy has been widely documented across current literature, 5 but is beyond the scope of this review.
Platinum therapies induce interstrand and intrastrand DNA adducts, which trigger mismatch repair (MMR) pathways and apoptosis. 6 Taxanes bind β-tubulin to prevent microtubule depolymerization, in turn, promoting cell cycle arrest and apoptosis. 7 Although 70% of OC patients initially respond to chemotherapy, subsequent chemoresistance and disease recurrence remain significant concerns and underpin 90% of eventual deaths. 8 For several decades, it has been well-documented that genetic mutations affecting a plethora of processes including DNA repair, epithelial-mesenchymal transition (EMT), and drug export, are implicated in chemoresistance. 9 Nevertheless, in some instances, it appears that genetic alterations alone may not fully elucidate chemoresistance. 10,11 Thus, an important role of epigenetic mechanisms has emerged. Since an initial association between DNA methylation, the most common epigenetic alteration, and human cancer was established in 1983, 12 the development of novel technologies based on methylation arrays (e.g., Illumina's HumanMethylation450k) and methylation sequencing (e.g., reduced representation bisulfite sequencing), 13 have facilitated a greater understanding of aberrant DNA methylation in OC chemoresistance.
DNA methylation is mediated by DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) which add methyl groups to cytosine residues of CpG dinucleotides. Tumor cells generally exhibit global DNA hypomethylation (particularly across repetitive DNA sequences), but de novo hypermethylation at CpG islands. This latter phenomenon often occurs at promoter sites of cancer-related genes, typically downregulating their expression. 14 Studies have shown that hypermethylation at CpG islands of tumor suppressor genes is associated with earlier disease recurrence and reduced progressionfree survival in OC patients. 15,16 Inspired by such findings, over the last two decades, various studies have highlighted a range of hypo/ hypermethylated candidate genes that are associated with OC chemoresistance ( Table 1).
Although methylation can occur at intergenic regions, this review will focus on methylation events occurring at the promoter sites of EOC resistance-associated genes. We will discuss and evaluate genes that are involved in well-established resistance-related processes/ pathways, including drug export (ATP-binding cassette subfamily B

| Methodology
A comprehensive search, based on our keywords, was conducted on the PubMed database and Google Scholar from February 2021 to August 2022. Our selected terms included "Ovarian cancer," "chemoresistance," "methylation," "epigenetics," and "DNA methyltransferase inhibitor." The literature search strategy for the data in tables and results is provided in Supporting Information: Figure S1.
The set inclusion criteria for selected articles were (a) original studies (including preclinical studies), observational and interventional studies, or randomized controlled trials; (b) articles with fulltext accessibility; (c) articles in the English language. We excluded articles that were (a) case reports, editorials, reviews, and commen-

| DNMTi-Evidence from clinical trials
Given that DNA hypermethylation, unlike genetic mutations, can be readily reversed, there has been significant therapeutic interest surrounding DNMTi (Figure 3). Azacytidine and decitabine were the decitabine-induced demethylation positively correlated with progression-free survival, 104 reinforcing the significance of their methylation profiles in chemoresistance. Nevertheless, a phase II trial involving single-dose decitabine preceding carboplatin treatment was prematurely terminated owing to a lack of efficacy and frequent hypersensitivity reactions and neutropenia. 110 This finding is elucidated by the fact that, unlike with prolonged low-dose DNMTi treatment, a single high-dose administration of DNMTi is unlikely to mediate prolonged demethylation but more likely to elicit doselimiting toxicities. 111 The recent emergence of a second-generation DNMTi, guadecitabine, could enhance the feasibility of combined DNMTi and platinum therapy. Guadecitabine is a dinucleotide of decitabine and deoxyguanosine that possess a longer half-life than decitabine/ azacytidine, 112 owing to its resistance to cytidine deaminasemediated degradation. In an initial phase I trial, guadecitabine preceded by carboplatin was tolerated and elicited an ORR of 15% among heavily pretreated patients. 106 The dose and schedule of combined guadecitabine and carboplatin treatment employed has since been validated by similar phase I studies based on germ cell cancer 113 and urothelial carcinoma. 112 Subsequently, a randomized phase II trial based on 100 platinumresistant OC patients receiving either guadecitabine and carboplatin (G + C) or a nonplatinum treatment of choice (TC) ensued. 107 Although the study did not reach the intended primary endpoint (median progression-free survival [PFS]), the 6-month PFS rate was significantly greater in the G + C cohort (37% compared to 11%).
Notably, this trial lacked a sole carboplatin treatment control group, thus it is difficult to ascertain to what extent guadecitabine was responsible for improved clinical outcomes.

| Future direction of DNMTi and methylated-based biomarkers
Although combined DNMTi and chemotherapy has shown promise,

| CONCLUSION
Here, we have discussed how epimutations across key protumorigenic processes, including drug export, apoptosis, and Wnt/β-catenin signaling, are linked with OC chemoresistance. Notably, methylation events that upregulate the expression of ABC transporters (e.g., ABCB1 and ABCG2) confer resistance through promoting chemotherapeutic drug efflux. Moreover, hypermethylation events that downregulate the expression of proapoptotic (e.g., RASSF1A) and/or DNA repair genes (e.g., MLH1, MSH2) are associated with chemoresistance in clinical OC samples. Aberrant methylation can also promote pro-oncogenic pathway signaling (e.g., Wnt/β-Catenin) through altering the expression of regulators of such processes (e.g., SFRPs, ZNF671). Finally, propelled by bioinformatic advances, some signaling proteins (e.g., NCALD), which have previously been associated with other cancers, are increasingly being seen in a new light; such genes could represent novel biomarkers, however, their specific functional/metabolic roles in OC remain largely unknown.
Although a myriad of clinically and statistically significant data associate these biomarkers with OC chemoresistance, there are still considerable gaps in our knowledge relating to the epimutations discussed (summarized in Table 3). Examples of largely unexplored domains include how biomarker expression varies prechemotherapy versus postchemotherapy (i.e., intrinsic vs. acquired resistance), disparities across OC histotypes, and the significance of the specific site of methylation within the promoter region(s). Given these limitations, it is unlikely that the maximal potential of these prognostic biomarkers will be realized without further work. Ideally, once these key domains have been Mara Artibani: Supervision; writing-review and editing.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Mara Artibani and Kaiyang Song had full access to all the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.

ETHICS STATEMENT
No animals or clinical samples were used in this study.

TRANSPARENCY STATEMENT
The lead author Mara Artibani affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.